A NUMB–EFA6B–ARF6 recycling route controls apically restricted cell protrusions and mesenchymal motility

International audienceThe endocytic protein NUMB has been implicated in the control of various polarized cellular processes, including the acquisition of mesenchymal migratory traits through molecular mechanisms that have only been partially defined. Here, we report that NUMB is a negative regulator of a specialized set of understudied, apically restricted, actin-based protrusions, the circular dorsal ruffles (CDRs), induced by either PDGF or HGF stimulation. Through its PTB domain, NUMB binds directly to an N-terminal NPLF motif of the ARF6 guanine nucleotide exchange factor, EFA6B, and promotes its exchange activity in vitro. In cells, a NUMB-EFA6B-ARF6 axis regulates the recycling of the actin regulatory cargo RAC1 and is critical for the formation of CDRs that mark the acquisition of a mesenchymal mode of motility. Consistently, loss of NUMB promotes HGF-induced cell migration and invasion. Thus, NUMB negatively controls membrane protrusions and the acquisition of mesenchymal migratory traits by modulating EFA6B-ARF6 activity

RAB11, une petite GTPase des endosomes de recyclage (étude de trois de ses partenaires et de son rôle dans la mélalogénèse)

Rab 11 est une GTPase principalement associée au compartiment endosomal de recyclage (ERC) où elle joue un rôle d'organisateur de ces membranes. Dans les cellules Mnt1, Rab11 colocalise partiellement avec la forme immature de Pmel17, une protéine de la matrice mélanosomale. La surexpression de Rab11wt et Q70L perturbe la distribution de la forme mature de Pmel17 et induit une diminution de la mélanine intracellulaire. Nous avons démontré que Rab11 joue un rôle dans la maturation de Pmel17, puisque le mutant Q70L induit une accumulation d'une de ses formes précurseurs. De plus, Rab11 interagit avec la queue cytoplasmique de Pmel17. Trois nouveaux partenaires de Rab11 ont été étudié. Le premier correspond à la chaîne Beta3B du complexe adaptateur AP-3. Nous avons démontré que les mélanocytes Mnt1 expriment la sous-unité Beta3B et qu'elle colocalise partiellement avec Rab11. A l'aide d'un essai in vitro, nous avons démontré que Rab11 intervient dans le recrutement du complexe AP3 neuronal sur les membranes du ERC. Nos données suggèrent un rôle de Rab11 dans la maturation de Pmel17 lors de son passage par des compartiments endosomaux de recyclage vers les structures mélanosomales. Au niveau du ERC, l'interaction Rab11/AP-3BetaB pourrait intervenir dans la ségrégation de Pmel17. Le deuxième partenaire correspond à RCP, une protéine interagissant avec Rab11 et Rab4. Nos résultats démontrent que RCP est une nouvelle protéine impliquée dans le recyclage endosomal. Enfin, le dernier partenaire correspond à la protéine Rab3lP, homologue des deux GEF Sec2p et GRAB et interagissant aussi avec Rab3. In vitro, nous avons mis en évidence une activité d'échange qui est pourtant plutôt faible. Néanmoins, in vivo, par des techniques de FRAP, nous avons obtenu des résultats qui confirment une activité GEF de Rab3IP sur Rab11. La surexpression de Rab3IP induit une redistribution intracellulaire du TfR analogue à celle induite par les formes mutantes constitutivement actives de Rab11.Rab11 is a small GTPase localized on endosomal recycling compartment (ERC) membranes and implicated in membrane organization and protein recycling to plasma membrane. The molecular mechanisms of membrane traffic leading to melanosome biogenesis are poorly understood. We demonstrate here that active mutated forms of Rab11 reduce the melanin content of melanocytes, while a corresponding Rab4 mutant does not. We show that this is mainly due to the inhibition of the processing and transport of Pmel17, a protein involved in the early stages of melanosome biogenesis. We also provide evidence that Rab11 directly binds to the cytoplasmic tail of Pmel17. Moreover, we found that the neurospecific BetaB subunit of the AP-3 coat complex is associated with Rab11 positive membranes in melanocytic cells and specifically interacts with Rab11. Our results constitute the first evidence that a Rab protein, through specific interactions with tissue-specific coat complex and cargo, regulates sorting from a compartment related to the ERC toward later stages of membrane maturation. Besides these results, were identified by two hybrid screens two novel Rab11 partners. The first, called RCP (Rab coupling protein) is an endosomal membrane-associated protein Interacting both with Rab11 and Rab4. We demonstrated that RCP is implicated in endosomal recycling. The second partner corresponds to Rab3IP, the human orthologue of rat Rabin 3, a previously identified Rab3A interacting protein with sequence homology to two Rab Guanine nucleotide Exchange Factors (Sec2p and GRAB). In vitro results indicate that Rab3lP has a weak exchange activity on Rab11. Furthermore, over-expression of GFP-tagged Rab3IP in HeLa cells induces a phenotype closely related to that generated by Rab11 dominant-positive mutant. Finally, using FRAP experiments, we were able to demonstrate that the fluorescence recovery of GFP-Rab11 after photobleaching of the pericentriolar area is affected in cell co-expressing YFP-Rab3IP.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

The Structural GDP/GTP Cycle of Rab11 Reveals a Novel Interface Involved in the Dynamics of Recycling Endosomes

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A Novel Golgi Membrane Protein Is a Partner of the ARF Exchange Factors Gea1p and Gea2p

The Sec7 domain guanine nucleotide exchange factors (GEFs) for the GTPase ARF are highly conserved regulators of membrane dynamics and protein trafficking. The interactions of large ARF GEFs with cellular membranes for localization and/or activation are likely to participate in regulated recruitment of ARF and effectors. However, these interactions remain largely unknown. Here we characterize Gmh1p, the first Golgi transmembrane-domain partner of any of the high-molecular-weight ARF-GEFs. Gmh1p is an evolutionarily conserved protein. We demonstrate molecular interaction between the yeast Gmh1p and the large ARF-GEFs Gea1p and Gea2p. This interaction involves a domain of Gea1p and Gea2p that is conserved in the eukaryotic orthologues of the Gea proteins. A single mutation in a conserved amino acid residue of this domain is sufficient to abrogate the interaction, whereas the overexpression of Gmh1p can compensate in vivo defects caused by mutations in this domain. We show that Gmh1p is an integral membrane protein that localizes to the early Golgi in yeast and in human HeLa cells and cycles through the ER. Hence, we propose that Gmh1p acts as a positive Golgi-membrane partner for Gea function. These results are of general interest given the evolutionary conservation of both ARF-GEFs and the Gmh proteins

The GTP/GDP Cycling of Rho GTPase TCL Is an Essential Regulator of the Early Endocytic Pathway

Rho GTPases are key regulators of actin dynamics. We report that the Rho GTPase TCL, which is closely related to Cdc42 and TC10, localizes to the plasma membrane and the early/sorting endosomes in HeLa cells, suggesting a role in the early endocytic pathway. Receptor-dependent internalization of transferrin (Tf) is unaffected by suppression of endogenous TCL by small interfering RNA treatment. However, Tf accumulates in Rab5-positive uncoated endocytic vesicles and fails to reach the early endosome antigen-1–positive early endosomal compartments and the pericentriolar recycling endosomes. Moreover, Tf release upon TCL knockdown is significantly slower. Conversely, in the presence of dominant active TCL, internalized Tf accumulates in early endosome antigen-1–positive early/sorting endosomes and not in perinuclear recycling endosomes. Tf recycles directly from the early/sorting endosomes and it is normally released by the cells. The same phenotype is generated by replacing the C terminus of dominant active Cdc42 and TC10 with that of TCL, indicating that all three proteins share downstream effector proteins. Thus, TCL is essential for clathrin-dependent endocytosed receptors to enter the early/sorting endosomes. Furthermore, the active GTPase favors direct recycling from early/sorting endosomes without accumulating in the perinuclear recycling endosomes

The EH network in <i>C. elegans</i>. An interaction diagram is shown representing <i>C. elegans</i> EH proteins (red circles) together with their interactors (blue circles); the interactors are further grouped into functional categories that were derived from the Wormbase and the Gene Ontology databases, from the literature, or inferred from functions of the mammalian homologues.

<p>Interactions uncovered in this study by Y2H are shown by light blue lines. Interactions confirmed by <i>in vitro</i> binding assays are shown by dark blue lines. Interactions not fully depending on the EH domain are shown with dashed lines. Additional interactions, derived from the BioGRID database (<a href="http://thebiogrid.org/" target="_blank">http://thebiogrid.org/</a>) and from the literature, are shown by red lines. The picture was initially generated using the Osprey software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056383#pone.0056383-Breitkreutz1" target="_blank">[96]</a> and then edited with Adobe Illustrator.</p

Yeast Two Hybrid analysis of EH-proteins in <i>C. elegans</i>.

<p>(A) Schematic diagram of the five EH-containing proteins in <i>C. elegans</i>. Note that several isoforms are reported in wormbase. Here, we show the isoforms cloned, sequenced and used for the described experiments. Baits used for the Y2H are indicated by black lines. For EHS-1, two distinct baits were used in the screens, since a bait spanning the three EH domains showed self-activation. CC, coiled-coil region; SH3, region containing multiple SH3s in ITSN-1; PxxP, region containing multiple SH3-binding sites in EHS-1; DPFs, region containing multiple AP-2-binding sites in EHS-1; P-loop, nucleotide-binding domain in RME-1. (B) Results of the Y2H screen. The 26 identified EH-interactors are listed. Potential EH-binding motifs are indicated. Black, interactions detected in the initial screen; gray, interactions detected in the re-transformation assay (see text). The number of clones identified in the initial screen is also shown. No interactions were detected for R10E11.6. (C) The indicated genes were tested by quantitative PCR in the yeast library used for the Y2H screening. The number of EH-interacting motifs (NPF) and the frequency of identification in the Y2H (H, high; In, intermediate; L, low; No, no interaction) are shown at the bottom. The estimated number of copies present in the cDNA library is shown, by grey bars, in arbitrary units relative to the level of representation of <i>epn</i>-1 that was set to 100. As a comparison we show, using black bars, the frequency of isolation of the various clones in Y2H, again relative to the frequency of isolation of <i>epn</i>-1 that was set to 100 ( = 45 clones).</p

A Snapshot of the Physical and Functional Wiring of the Eps15 Homology Domain Network in the Nematode

<div><p>Protein interaction modules coordinate the connections within and the activity of intracellular signaling networks. The Eps15 Homology (EH) module, a protein-protein interaction domain that is a key feature of the EH-network, was originally identified in a few proteins involved in endocytosis and vesicle trafficking, and has subsequently also been implicated in actin reorganization, nuclear shuttling, and DNA repair. Here we report an extensive characterization of the physical connections and of the functional wirings of the EH-network in the nematode. Our data show that one of the major physiological roles of the EH-network is in neurotransmission. In addition, we found that the proteins of the network intersect, and possibly coordinate, a number of “territories” of cellular activity including endocytosis/recycling/vesicle transport, actin dynamics, general metabolism and signal transduction, ubiquitination/degradation of proteins, DNA replication/repair, and miRNA biogenesis and processing.</p> </div

In vitro binding assays.

<p>Sixteen interactors, identified by Y2H (listed at the bottom), were expressed as GST-fusion proteins and used for <i>in vitro</i> binding assays with FLAG-EH proteins expressed in Phoenix cells. Results are the average of three independent experiments (examples are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056383#pone.0056383.s003" target="_blank">Figure S3</a>), and are expressed in arbitrary units on a scale 0–100, in which 100 represents the efficiency of the pull-down for the strongest interacting protein in each panel.</p